In the development of biosensors for the early detection of disease, the availability of high specificity and affinity ligands for biomarkers that are indicative of a pathogenic process is important. Biopanning of phage displayed peptide libraries on intact cells has proven to be a successful route for the identification of cell-specific ligands. The peptides selected from these combinatorial libraries are often able to distinguish between diseased cells and their normal counterparts as well as cells in different activation states. These ligands are small and chemical methodologies are available for their regiospecific derivatization. As such, they can be incorporated into a variety of different diagnostic and therapeutic platforms. However, current methods for generating monoclonal antibodies against cell surface expressed antigens are inefficient and time-consuming, requiring the overproduction and/or purification of each extracellular target of interest. Cell surface proteins are dynamically regulated and their expression profiles can change depending on cellular stress or disease states.
Although there has been considerable effort spent on optimizing in vitro phage and yeast display techniques, selection methods using large antibody libraries or polypeptide libraries against these membrane-bound targets on whole cells pose significant problems. Major limitations remain in the existing art, such as distinguishing specific and non specific antibody-antigen complexes, wherein the antigen is the cell surface protein of interest (POI), isolation from the mixture of antibodies, directing antibodies towards the protein of interest or to the cell surface or family of proteins of interest (FPOI), and binding of antibodies to the background protein on the cell surface.
The general approach to remove antibodies or polypeptides bound to background protein comprises steps of depleting phage-antibody or phage-polypeptide libraries that are bound to background proteins and then adding those surviving phage to cells containing the POI. This approach is cumbersome and requires multiple selections. Another major limitation of generating and isolating antibodies or polypeptides to a specific protein of interest is the over-expression of the protein of interest; it is known that over-expression can upregulate other proteins not related to the POI and increase background antibodies, thereby complicating antibody-POI complex selection and increasing the non-specific binding. Thus, alternate methods for selection of antibodies that bind to protein of interests are needed.